A SUB-ACUTE STUDY TO EVALUA TE THE USE OF ACUTE PHASE PROTEINS IN MALLARD DUCKS
(Anas platyrhynchos ), AS INDICATORS OF
LONG-TERM HEALTH AFTER INGESTION OF CRUDE OILTHESIS PRESENTED AS PARTIAL REQUIREMENT OF DOCTORATE IN BIOLOGY BY LYNN MILLER SEPTEMBER 2014
Avertissement
La diffusion de cette thèse se fait dans le respect des droits de son auteur, qui a signé le formulaire Autorisation de reproduire et de diffuser un travail de recherche de cycles supérieurs (SDU-522 - Rév.01-2006). Cette autorisation stipule que «conformément à l'article 11 du Règlement no 8 des études de cycles supérieurs, [l'auteur] concède à l'Université du Québec à Montréal une licence non exclusive d'utilisation et de publication de la totalité ou d'une partie importante de [son] travail de recherche pour des fins pédagogiques et non commerciales. Plus précisément, [l'auteur] autorise l'Université du Québec à Montréal à reproduire, diffuser, prêter, distribuer ou vendre des copies de [son] travail de recherche à des fins non commerciales sur quelque support que ce soit, y compris l'Internet. Cette licence et cette autorisation n'entraînent pas une renonciation de [la] part [de l'auteur] à [ses] droits moraux ni à [ses] droits de propriété intellectuelle. Sauf entente contraire, [l'auteur] conserve la liberté de diffuser et de commercialiser ou non ce travail dont [il] possède un exemplaire.»
ÉTUDE SOUS-AIGÜE POUR ÉVALUER L'UTILISATION DES PROTÉINES DE PHASE AIGUË
CHEZ LES CANARDS COL VERTS (Anas platyrhynchos), COMME INDICATEURS DE LA SANTÉ À LONG TERME APRÈS L'INGESTION DE
PÉTROLE BRUT
THÈSE PRÉSENTÉE
COMME EXIGENCE PARTIELLE DU DOCTORAT EN BIOLOGIE
PAR LYNN MILLER
Les déversements d'hydrocarbures sont dévastateurs pour l'environnement à plusieurs niveaux. Plusieurs facteurs entrent en ligne de compte, particulièrement concernant la faune. L'effort de réhabilitation nécessite une expertise, des installations et de nombreuses personnes qui souhaitent gérer tous les aspects de la réponse. Tous les oiseaux qui ont été capturés ont été évalués et admis au processus de réadaptation. Le problème s'est posé lorsque ces animaux sont retournés à l'état sauvage. Sharpe (1996) a attiré l'attention sur l'apparence du faible taux de survie qui suit la réhabilitation de plusieurs espèces d'oiseaux. Si, en tant que rééducateurs, nous voulons vraiment faire le travail qui nous intéresse, la simple survie n'est pas la réponse à tout ce processus. La faune que nous nettoyons et que nous réhabilitons doit être en mesure de retourner à sa vie et de pouvoir vivre comme des animaux sauvages, ce qui incluse le fait de donner naissance à une progéniture en santé. Ce projet de recherche est axé sur le développement d'un outil de triage additionnel qui sera utile au cours de la phase précoce lors d'un déversement de pétrole. Il faut d'abord explorer l'évolution de la réponse des animaux en phase aigüe à travers les profils protéiques, et lier ensuite ce processus biochimique au résultat à long terme. C'est avec un certain soulagement teinté de tristesse que je peux affirmer que cela a été un projet de recherche qui a très bien réussi. L'électrophorèse des protéines peut être utilisée comme outil pour guider l'effort de redressement. Il reste évidemment beaucoup de travail à faire. Nous avons besoin des électrophérograrnmes de base et de la· quantification des groupes de protéines trouvées dans ma technique pour de nombreux oiseaux aquatiques généralement touchés. Donc les oiseaux admis à un nettoyage lors des opérations de déversement, pourront bénéficier de mes recherches. Cette prestation peut être l'euthanasie, mais il y aura une approche beaucoup plus humanitaire pour venir en aide aux oiseaux sur le plan toxicologique.
L'entreprise de cette recherche prend de multiples facettes, avec en premier lieu, l'installation et les oiseaux. J'ai eu la chance de pouvoir faire ce travail à la station Delta Waterfowl Research, Delta Beach, au Manitoba. J'ai fait l'aller-retour pendant quatre ans, passant mes étés (et un hiver) auprès du troupeau de canards à mener mes recherches. J'ai travaillé tous les aspects du projet à partir de la prise en charge du troupeau de canards Delta, à la gestion d'une installation spécialisée, la supervision des techniciens d'été, et toutes les activités quotidiennes liées à ma recherche. Je n'aurais pas pu réaliser ce travail sans l'équipe de la station de la sauvagine de recherche Delta. L'installation est de classe mondiale et les connaissances que j'ai acquises auprès des spécialistes travaillant à la station sont immenses.
Pour m'avoir aidée à explorer l'électrophorèse capillaire des protéines capillaires comme offrant le meilleur outil potentiel pour ce projet, je suis reconnaissante de l'appui technique que j'ai reçu de la part de Sylvie Lernieux de l'UQAM. J'ai pu profiter de cette base pour le laboratoire du Dr Cameron Skinner à l'Université Concordia, de raffmer et de l'utiliser pour explorer les protéines exprimées par mes canards tout au long de la période de recherche dans son ensemble. Dr Skinner et son équipe m'ont apporté une aide précieuse et inestimable.
J'ai pu envoyer des échantillons au Dr GTégory Bédécarrats au Département des sciences animales et avicoles de l'Université de Guelph en Ontario. ll a gracieusement réalisé en utilisant sa radio-irnmuno essai pour arriver à confirmer qu'il y a un changement sous-jacent dans le cycle de prolactine des colverts femelles qui reçoivent une dose d'hydrocarbure élevée.
Un grand nombre de personnes ont manifesté leur soutien et leur confiance dans mon travail. Je tiens à remercier tout particulièrement mon mari, Michel Béland. ll m'a
soutenue dans tous les aspects de cette recherche; finances, expertise informatique, soutien moral, et il a été à mes côtés lors des hauts et des bas qu'engendre la recherche - je vous remercie. Mes superviseurs, qui n'ont jamais montré de découragement, ont été à mes côtés pendant toutes ces années à m'aider à donner vie à ma passion - je vous remercie, Phil et Diana. J'ai également reçu le soutien financier de la Fondation Delta Sauvagine, de la Société québécoise pour la protection des oiseaux, le Oiled Wildlife Care Network en Californie, ainsi que le Dr Philip Spear. Merci.
Plusieurs personnes ont manifesté un intérêt particulier et m'ont grandement aidé pendant toutes ces années: ma fille, Danielle Miller Béland, M. Phil Kline (décédé), Michel Boisvert, Shirley Desrosiers et le Dr Charlie Blurnmer, pour n'en nommer que quelques-uns. Et je tiens à souligner la précieuse contribution Patricia Hennesey, qui a repris ma thèse pour m'aider à l'améliorer, ce que je ne n'aurais pu accomplir sans son soutien et son dévouement.
FOREWORD
Oil spills are devastating to the environment at many levels. There are many factors that are involved in an oil spill response, especially when wildlife is involved. The rehabilitation effort requires expertise, facilities and many willing people to manage all the aspects of the response. AU captured birds are evaluated and admitted to the rehabilitation process. The problem arises once these animais are returned to the wild. Sharpe (1996) focused attention on the apparent poor post rehabilitation survival in many bird species. If we as rehabilitators are truly doing the job we care about, for birds to simply survive is not the answer to this whole process. The wildlife that we clean and rehabilitate must be able to return to their lives and continue as truly wild animais, which includes producing normal healthy off-spring. This research project focused on developing a new triage tool to aid in the early phase of the oil spill response. First, by exploring the evolution of the animal's acute phase response through the protein profiles, and then by linking this biochemical process to the long term outcome. It is with relief tinged with sadness that I can say this has been a very successful research project. Protein electrophoresis can be used as a tool to guide the recovery effort. Of course there is still work to do; we need the baseline electropherograms and the quantification of the protein groups found using my technique for many of the commonly impacted waterfowl. The birds then admitted to a spill 'clean-up' operation can benefit from my research. That benefit may be euthanasia, but it will be a far more humane approach to toxicologically damaged birds.
To undertake this research required many facets. First was the facility and birds. I was very fortunate to be able to do this work at Delta Waterfowl Research Station, Delta Beach, Manitoba. For four years, I commuted, spending summers (and one
winter) there, conducting my research with the Delta Waterfowl Foundation duck flock. I worked all aspects of the project from the care taking of the flock, to managing a dedicated facility, supervising the summer technicians, and all daily activities related to my research. I could not have done this work without the Delta Waterfowl Research station team. The facility is world class and the knowledge I gained by being able to turn to the people at the station is imrneasurable.
To explore whether protein capillary electrophoresis can offer the best potential tool for this project, I am grateful to the technical support I gained at UQAM from Ms Sylvie Lemieux. I was able to take this base knowledge to Dr Cameron Skinner's lab at Concordia University, refme and use it to explore the proteins expressed by my ducks throughout the who le research period. Dr Skinner and his team' s help were invaluable.
I was able to send specimens to Dr Grégory Bédécarrats, Animal and Poultry Science Departrnent, University of Guelph, Ontario. He graciously performed prolactin radio-immuno assays to help confmn that there is an underlying shift in the cycle of prolactin in the high oil dose female mallards.
There are so many people who have shown their support and faith in my work. I want to especially thank my husband, Michel Béland. He has supported me in all aspects of this research; fmancially, computer expertise, moral support, and has been there through the many ups and downs that research engenders - thank you. My supervisors, never despaired openly, were there for me through these years of trying to bring my passion to life - thank you Phil and Diana. I have also had fmancial support from Delta Waterfowl Foundation, the province of Québec's Society for the Protection of Birds, Oiled Wildlife Care Network in California and Dr Philip Spear. Thank you.
There are also so many people who have taken special interest and have helped enormously through these years; my daughter Danielle Miller Béland, Mr. Phil Kline ( deceased), Michel Boisvert, Shirley Desrosiers and Dr. Charlie Blummer to name a few. Finally, Patricia Hennesey has taken my thesis and worked with me to irnprove it tremendously; I could not have done it without her dedicated help.
LISTE DES FIGURES ... xv
LISTE DES TABLEAUX ... xxiii
RÉSUMÉ ... xxvii
INTRODUCTION ... 1
CHAPITRE I LITERA TURE REVIEW ... 4
1.1. Historical background about oil spills ... 5
1.1.1. The first ail tanker spill.. ... 5
1.1.2. The media response to the plight of oiled birds ... 6
1.1.3. Oil res panses -the Canadian situation ... 7
1.2. Oil impacts on birds and the rehabilitation effort ... 8
1.2.1. Background ... 8
1.2.2. Development of a professional wildlife rehabilitation response to ail spills ... 10
1.2.3. Survival data in birds following ail spill exposure ... 13
1.2.4. Oil toxicity in birds ... 14
1.2.5. Impacts of ail spills in birds at the population level ... 15
1.2.6. Impacts of ail spills on reproduction in birds ... 16
1.2. 7. Immune function impacts in oiled birds ... 17
1.2.8. Impacts of ail in young birds ... 18
1.2.9. Limitations to oiled bird rehabilitation ... 18
1.3 .1. An overview of the acute phase response and the subsequent 1.3.2. 1.3.3. 1.3.4. 1.3.5. 1.3.6. 1.3.7. 1.3.8. 1.3.9. 1.3.10. 1.4. 1.4.1. 1.4.2.
induction of acute phase pro teins ... 20
An overview of the cytokine response ... 23
An overview of the acute phase pro teins ... 25
Acute phase pro teins in hum ans ... 29
The use of serum prote in electrophoresis in veterinary practice ... 32
Acute phase response in other vertebrate species ... 3 8 Acute phase pro teins in response to a toxic event ... 41
Acute versus chronic changes in acute phase proteins ... 45
Perspectives for use of acute phase proteins as biomarkers in oil spill responses ... 4 7 The use of capillary electrophoresis (CE) in avian research ... .47
The avian model -the mallard and its annual cycle ... 48
Introduction ... 48
August- September ... 50
1.4.2.1. Location ofbirds ... 50
1.4.2.2. Reproductive cycle ... 50
1.4.2.3. Moult. ... 51
1.4.2.4. Onset of Breeding Behaviour ... 52
1.4.3. October-November ... 55 1.4.3.1. Location ofbirds ... 55 1.4.3.2. Reproductive cycle ... 56 1.4.4. December to January ... 57 1.4.4.1. Locationofbirds ... 57 1.4.4.2. Reproductive cycle ... 57 1.4.4.3. Moult ... 57 1.4.5. February-April. ... 58 1.4.5.1. Location ofbirds ... 58
1.4.5.2. Reproductive cycle ... 59 1.4.6. April -May ... 59 1.4.6.1. Reproductive cycle ... 59 1.4.7. June-July ... oo···.-···0062 1.4.7.1. Location ofbirds ... oo ... 62 1.4.7.2. Moult. ... 63 1.4.7.3. Reproductive cycle ... 63 1.4.8. Summary ... 64
1.5. Hypotheses and Objectives ... 64
CHAPITRE II METHODS AND MA TERIALS ... 000000 ... 67
2.1. Animal housing facility ... 68
2.1.1. The breeding pens ... 00 ... 00 .. 00 00 .. 00 .. 69
2.1.2. The outdoor a vi aries ... 00 ... 71
2.1.3. The indoor a vi aries ... 73
2.1.4. The blood collection area ... 75
2.1.5. The nest boxes ... 7 5 2.1.6. Artificial incubation and egg care ... 76
2.2. Establishing the oil dose groups ... 79
2.3. Housing of the groups throughout the year (Table 2.1 ) ... 81
2.3.1. July and August ... 81
2.3.2. September ... 81
2.3.3. October to earl y April ... 82
2.3.4. April and May ... 82
2.3.5. May and June ... 00 ... 00 .. 00 ... 83
2.3.6. July to September ... 83
2.4.1. Eggs ... 84
2.4.2. Ducklings ... 88
2.5. The Test Oil ... 89
2.6. Dose rates and dosing process ... 93
2.7. Blood sampling ... 94
2.8. Euthanasia and tissue preparation ... 97
2.9. Capillary electrophoresis ... 98
2.1 O. Behavior ... 109
2.10.1. Pair bonding ... 109
2.10.2. Second clutch attempt ... 109
2.10.3. Nest boxes ... 110
2.1 0.4. Blood sampling issues ... · ... 111
2.11. Prolactin determination ... 113 2.12. Statistics ... 113 CHAPITRE III RESULTS ... 115 3.1. Behavior ... 116 3.1.1. Agitation ... 117
3.1.2. Pair bon ding ... 119
3.1.3. Nest construction ... 121
3.2. Eggs ... 125
3.2.1. Egg production ... 125
3.2.2. Egg fertility ... 127
3.2.3. Female dose group egg data ... 127
3.2.4. Male dose group egg data ... 130
3.3. Prolactin ... 13 3 3.4. Protein profiles ... 135
3.4.1. The acute phase: days one to day five post oil dosing ... 137
3.4.2. The transitional phase; from sixto fifteen days post oil dosing ... 158
3.4.3. The chronic phase; after sixteen da ys post oil dosing ... 160
3.4.4. Days 19 and 21 after oil dosing in male mallards ... 160
3.4.5. Day 72 after oil dosing in female mallards ... 167
3.4.6. Day 278 after oil dosing in female mallards ... 172
3.4.7. Day 334 after oil dosing in male mallards ... 175
3.4.8. Day 414 after oil dosing in female mallards ... 179
3.5. Routine blood parameters and gross autopsy results ... 184
CHAPITRE IV DISCUSSION .. , ... 185
4.1. Behaviour ... 186
4.1.1. Agitation ... 186
4.1.2. Pair bon ding ... 190
4.1.3. Nest construction ... 193
4.1.4. Egg production ... 194
4.2. Prolactin ... 200
4.3. Protein electrophoresis ... 203
4.3.1. Introduction ... 203
4.3.2. The acute phase: da ys one to day five post oil dosing ... 204
4.3.3. The transitional phase; from sixto fifteen days post oil dosing ... 206
4.3.4. The chronic phase; after sixteen da ys post oil dosing ... 207
4.3.5. Pro teins -the bottom line ... 209
CONCLUSION ... 210
5.1. CONCLUSION ... 211
APPENDICE A
OIL SPILL REHABILITA TI ON RESPONSE FOR BIRDS ... 222
A. Introduction ... 223
A.1 Capture ... 224
A.1.1 The reality of cap turing oiled birds ... 224
A.1.2 The Deepwater Horizon situation ... 225
A.2 Admission ... 226
A.2.1 Overview ... 226
A.2.2 Deepwater Horizon admission pro cess ... 226
A.2.3 Blood sampling and tests ... 229
A.2.4 Stabilization process ... 233
A.3 Housing ... · ... 234
A.4 Feeding and monitoring intake ... 236
A.5 Human health and safety ... 237
A.6 Stabilization ... 240
A. 7 The wash line ... 241
A. 7.1 Preparation ... 241
A.7.2 Washing birds ... 242
A.8 The final steps ... 24 7 A.9 Release ... 248
APPENDICEB
STATISTICAL EVALUATION OF SERUM PROTEIN DATA ... 249
Figures Page
1.1 The front page of the National Geographie for J anuary, 1990 ... 6 1.2 The number of days for bands to be recovered between non-oiled birds
(control) vs. oiled, rehabilitated and released birds ( adapted from Sharpe, 1996) ... 13 1.3 An overview of the acute phase response in an organism (Heinrich et al,.
1990) ... 21 1.4 Schematic representation of the processes involved in the induction of
acute phase proteins by hepatocytes (adaptedfrom Heinrich et al., 1990, in Gruys et al., 1994) ... 22 1.5 Interleukin (IL)-6 concentration in the sera of chickens inoculated with
Escherichia coli lipopolysaccharide (LPS) and PBS (control). The asterisks designate that the levels of LPS group were significantly (P < 0.05) higher than those of the PBS group at indicated time points. Vertical bars represent the SD. (from N akamura et al., 1998) ... 24 1.6 The acute phase protein al-acid g1ycoprotein (al-AG) concentration in
the sera of chickens inoculated with Escherichia coli lipopolysaccharide
(LPS) and PBS (control). The asterisks designate that the levels of LPS
group were significantly (P < 0.05) higher than those of the PBS group at indicated time-points. Vertical bars represent the SD. (from Nakamura et al., 1998) ... 25 1. 7 Generalized patterns of change in plasma concentrations of sorne acute
phase proteins in response to a moderate in:flarnmatory stimulus in humans (Gabay and Kushner, 1999) ... 26
1.8 A representative normal electropherogram for a psittacine (from Cray, 1995) ... 35
1.9 Representative plasma protein electrophoretograms of the loggerhead sea turtle, Caretta caretta. Image (1) The dark verticallines from le ft to right denote the breaks between the albumin (A), Alpha globulin (B), Beta globulin (C), and Gamma globulin (D) fractions. Image (2) with a pre-albumin (P) band (adaptedfrom Gicking et al., 2004) ... 39
1.10 Box plots comparing the distribution of: (a) serum amyloid A (SAA); (b)
haptoglobin; (c) a1-acid glycoprotein in 31 cattle . with acute
inflammation and 50 cattle with chronic inflammation. The median is marked with a line, the box shows the 251h to 75th percentile, the
whiskers show the 1 oth and 901h percentiles and outliers are marked with
closed circles (adaptedfrom Horadagoda et al., 1999) ... 46
1.11 A generalized scheme showing the transduction of environmental
factors into neuroendocrine and endocrine secretions which regulate waterfowl reproduction and associated factors (Bluhm, 1992) ... 49
1.12 The male (right) and female (left) mallard in altemate or breeding
plumage ... 52
1.13 Ten courtship poses which belong to the common genetic heritage of
surface-feeding ducks are shown here as exemplified in the mallard: (1) initial bill-shake, (2) head-flick, (3) tail-shake, (4) grunt-whistle, (5) head-up - tail-up, (6) turn towards the female, (7) nod-swirnming, (8) turning the back of the head, (9) bridling, (10) down-up (Lorenz, 1958) ... 53
1.14 Relation of approximate period of pair bond formation in mallards to the
relative frequencies of major male displays. Adapted from Johnsgard
(1960) ... 54
1.15 Plasma luteinizing hormone (LH) levels (ng/ml) in female mallards and
ovarian weight (gm), month by month in 1971 and 1974 (adaptedfrom
Donham, 1979) ... 60
2.1 Diagram of the breeding pens showing the direction of water flow
(arrows) and the dose group layout. ... 70 2.2 Duck breeding pens at Delta Waterfowl Research Station ... 71
2.3 Outdoor aviary at Delta Waterfowl Research Station ... 72
2.4 Indoor aviary showing two of the four pens used to house waterfowl over the win ter at Delta Waterfowl Research Station ... 7 4 2.5 The deep water channel used along the sides of ail indoor aviaries ... 74
2.6. Duck nesting box designed by Ward and Batt (1973) ... 76
2.7 Humidaire® incubator for the artificial incubation of eggs ... 77
2.8 Hatching unit ... 78
2.9 Toe tag clipped into the foot web to identify individual ducklings ... 79
2.10 Images for days 6, 12 and 18, used in candling for the evaluation of embryological development in mallard eggs. (Ward and Batt, 1973) ... 85
2.11 Photographie index to determine the age of mallard embryos. The line below the age designation represents 1 cm (from Caldwell and Snart, 1974) ... 86
2.12. Toe tagged newly hatched mallard duckling ... 88
2.13 Blood sampling mallards, using the brachial wing vein ... 95
2.14 Trace of a normal mallard serum CE with peaks and their regions numbered 1 - 10 in Table 2.4. The tirne for the passage of the protein through the capillary is noted in seconds (s) ... 104
2.15 A normal human serum capillary electropherogram with the major protein peaks identified (Gay-Bellile et al., 2003) . ... 105
2.16 Example of plasma gel electrophoresis patterns of a Bar-headed Goose (Anser indicus). The major peaks noted are, (left to right) pre-albumin, albumin, al, a2, ~,y globulins (from Roman et al, 2009) ... 106
2.17 Example of plasma gel electrophoresis patterns of a Black Ki te (Milvus migrans). Image A shows the electrophoretic pattern using agarose gel electrophoresis. Image B shows the pattern using capillary zone electrophoresis with the pattern normalized to that of the standard gel electropherogram. The major peaks noted are, (left to right) pre-albumin, pre-albumin, and cd, a2, ~'and y globulins (adapted from Roman et al., 2013) ... 107 3 .1. Male mallard agitation levels at 14 da ys post oil dosing (number of
venepuncture attempts). There is a significant difference between the control dose group and the high oil dose. (Kruskal-Wallis; one way ANOVA; (P
=
<0.001) ... 117 3.2. Male mallard agitation levels at 334 days post oil dosing (number ofvenepuncture attempts) ... 118 3.3 The female mallard agitation levels at 414 days post oil dosing (number
of venepuncture attempts ) ... 119 3.4. Pair bonded vs. non-pair bonded female mallards in May 2002 show
significant differences between the expected pair bonding levels of the control hens compared to the pair bonding levels of the medium and high dose birds (chi-squared
= 18.94;
df=3; P=
<0.001) ... 120 3.5 Pair bonded vs. non-pair bonded male mallards in May, 2002 showsignificant differences between the expected pair bonding levels of the control males and the pair bonding levels of the low, medium and high dose birds (chi-squared df= 3: P
=
< 0,002) ... 121 3.6. Control dose female nest box with a straw bowl lined with downplucked from the hen's breast area. (Note: All adult birds at the Delta Waterfowl facility were nasal tagged with colour-coded plastic markers. These posed no problem to the birds and were well tolerated by all birds.) ... 123 3.7. High dose female nest box showing six eggs laid on a thin bed of straw.
Note that one egg was broken by the hen ... 124 3.8. Prolactin levels from female oil dose groups at 414 days post oïl dosing ... 134
3.9. The protein groups identified by capillary electrophoresis (CE) and nurnbered to indicate the individual groups evaluated. The peaks are measured in absorbance units (AU) over elution time in seconds (s). The insert guide shows the peak nurnber and the putative protein group .... 136 3.1 O. Gamma globulin (CE peak # 1) levels on Da ys 1 and 2 (poo led, da ys are
aNS effect) after oil dosing in both males and female mallards ... 138 3.11. Alpha 2 globulins (CE peak #5) on Days 1 and 2 after oil dosing in both
males and female mallards ... 139 3.12. Alburnin (CE peak #7) levels on Days 1 and 2 after oil dosing in both
males and female mallards ... 140 3.13. Transthyretin (CE peak #8) on Da ys 1 and 2 after oil dosing in both
males and female mallards ... 141 3.14. Protein peak #9 on Days 1 and 2 after oil dosing in both males and
female mallards ... 142 3.15. Peak # 10 on da ys 1 and 2 after oil dosing in both males and fern ale
mallards ... 143 3 .16. Protein peak #2 beta-globulin levels on da ys 3 and 4 after oil dosing in
female mallards ... 152 3.17 Protein peak #3 beta-globulin levels on days 3 and 4 after oil dosing in
female mallards ... 153 3.18. Protein peak #4 beta-globulin levels on days 3 and 4 (pooled) after oil
dosing in female mallards ... 154 3.19. Gamma globulin prote in peak levels on poo led data from da ys 19 and
21 after oil dosing in male mallards ... 161 3.20. Protein peak #2 beta globulin levels from days 19 and 21 after oil
3.21 Protein peak #4 beta globulin levels on the pooled data from days 19 and 21 after oil dosing in male mallards ... 163 3.22. Protein peak alpha 2 globulins levels on pooled data from days 19 and
21 after oil dosing in male mallards ... 164 3.23. Prote in peak transthyretin levels on da ys 19 and 21 after oil dosing in
male mal lards ... 165 3.24. Protein peak #2 beta-globulin levels on day 72 after oil dosing in female
mallards ... 168 3.25 Protein peak #3 beta-globulin levels on day 72 after oil dosing in female
mallards ... 169 3.26. Albumin protein peak levels on day 72 after oil dosing in female
mallards ... 170
3.27. Protein peak #2 beta-globulin levels on day 278 after oil dosing in
female mallards ... 173 3.28. Protein peak #3 beta-globulin levels on day 334 after oil dosing in male
mallards ... 175 3.29. Transthyretin protein peak levels on day 334 after oil dosing in male
mallards ... 1 7 6 3.30. Gamma globulin levels at 414 days after oil dosing in female mallards ... 180 3.31. Protein peak #3 in the beta-globulins at day 414 after oil doing in female
mallards ... 181 3.32. Unknown protein peak #9 in female mallards 414 days after oil dosing ... 182
A.1 Admission process at Fort George, LA. Dr. Erica Miller (right) from
Tri-State Bird Rescue and assistant examine a brown pelican
Note the protective clothing wom at all times when handling oiled birds in this event ... 228 A.2 All body surfaces were checked and records kept about the extent of
oiling on each admission, as can be seen on this brown pelican ... 229 A.3 Blood sample being collected from the transverse medial metatarsal
vein in a brown pelican ... 230 A.4 Hematocrit reader used to evalua te the percentage of red blood cells in a
blood volume ... 231 A.S Hand held refractometer used to quantify total solids which are
generally equivalent to total prote in levels ... 232 A.6 Scale used in the refractometer. The putative protein scale on the right
side is in grnJlOOrnl ... 233 A.7 A crate set up for smaller species needing intensive care. Food
appropriate to the species is provided. The red colouration is from heating lamps ... 235 A.8 A laughing gull (Larus atricilla) chick, covered in oil. Note the leg
band for identification ... 236 A.9 Daily weights were recorded. Two members on that team are pictured
here placing a pelican in the plastic container, tared and ready, on the scale ... 237 A.l 0 Full safety gear required for the handling of oiled birds ... 23 8 A.ll To help dissipate heat from around the body, slits were allowed in the
backs of the Tyvek® suits ... 239 A.l2 Joins between the gloves and suits were sealed using duct tape. The
A.13 Safety glasses were required whenever handling any birds as seen here as a handler carries a brown pelican ... 240 A.l4 The wash line at Fort George, LA ... 243 A.15 The rinse tearn at Fort George, LA ... 244 A.16 Water beads on the head feathers of a northem gannet (Morus bassinus) .
... 245 A.17 The drying room at Fort George, LA. Rehydration fluids are always kept
available to support any bird in distress through this process. The arrow indicates the fluids held in readiness for any bird in distress ... 246
Table Page
1.1 A summary of pathological fmdings reported in oiled birds submitted for pathological examination ... 9 1.2 Oiled Wildlife Care Network spill responses in which more than 25 live
birds were recovered and rehabilitated (Newman et al., 2003) ... 12 1.3 Acute phase proteins in humans and rats in response to a stressor
(Heinrich et al., 1990) ... 28 1.4 Major protein patterns (Albumin [Alb], al, a2, ~1, ~2 and y) with
diagnosis in human medicine (Keren, 1988) ... 30 1.5 Characteristics of selected acute phase reactants (from Johnson et al.,
1999) ... 31 1.6 The biological function of the acute phase proteins used in veterinary
medicine (Ceron et al., 2005) . .... 34 1. 7 Protein fraction changes in psittacine disease (jrom Cray and Bossard,
1995) ... 36 1.8 The fractions where major acute phase proteins are found in avian
plasma electrophoresis (adapted from Cray, 1997) with their overall biological function ... 3 7 1.9 Plasma protein fractions identified in loggerhead sea turtles, Caretta
caretta (jrom Gicking et al., 2004 ) .... 40
2.1 Housing, time frame and research activities ... 69
2.2 The randomly selected ducks were randomly assigned to a dose group and then to a sub-group ... 80
2.3 Excerpted data from ESD Brent Crude publication (1996, 1997) showing the Volatile Organic Compounds (VOC's) and their evaporation percentage loss and ppm in a closed system. This is then related to the time estirnated for this event under field conditions which may be highly variable ... 92 2.4 The oil dose ~er bird volumes and the total volume of Brent Crude Oil
and Tropican ... 93 2.5 Factors that differentiate standard Gel Electrophoresis from Capillary
Zone Electrophoresis developed in consultation with Dr. C. Skinner, Biochemistry Dept., Concordia University, Montreal ... 103 2.6 The protein groups corresponding to the protein peaks in Figure 2.14 ... 104 2. 7 Scoring system used to quantify agitation lev el in mallards during the
venepuncture ... 112 3.1. Nest boxes in the individual breeding pens where the hens had
attempted to main tain a straw based structure ... 122 3.2. Female mallard dose series total egg production in the indoor aviaries in
2002 ... 126 3.3. Egg production from clean female mallards housed with the male
mallard dose series in 2003 ... 126 3.4 Tabulated data for egg fertility data collected from male oil dose groups.
Unless otherwise noted, the data show the percentage of eggs with the number in (). Data sets with statistically significant differences are denoted with the same letter ... 129 3.5. Tabulated data for egg fertility data collected from male oil dose groups.
Unless otherwise noted, the data show the percentage of eggs with the number in (). Data sets with statistically significant differences are denoted with the same letter. ... 132
3.6. Female dose group protein ranges at days 1 and 2 post dosing. Bold typeset indicates significant differences. The numbers are shown as a percentage ofthe total ofthe peaks quantified ... 145 3. 7. Male dose group prote in ranges at da ys 1 and 2 post dosing. Bold
typeset indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 148 3.8. Sumrnary of probabilities and the tests used in exploring the differences
between male and female data points at days 1 and 2 post oil dosing. Welch's test was used in cases ofunequal variance ... 150 3.9. Testing for the effects of dose and sex as well as the interactions
between sex-dose on serum protein peaks at days 1 and 2 post oil dosing ... 151 3.10. Testing for the effects of dose and sex as well as the interactions
between sex-dose on serum protein peaks at days 1 and 2 post oil dosing ... 155 3.11. Female dose group protein ranges at days 3, 4 post dosing. Bold typeset
indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 157 3.12. Male oil dose group protein ranges at days 6 and 7 post dosing. Bold
typeset indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 159 3.13. Male dose group protein ranges at days 19 and 21 post dosing. Bold
typeset indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 166 3.14 Female dose group protein ranges at day 72 post dosing. Bold typeset
indicates significant differences. The numbers . are shown as a percentage ofthe total ofthe peaks quantified ... 171 3.15. Female dose group protein ranges at day 278 post dosing. Bold typeset
indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 174
3.16. Male dose group protein ranges at day 334 post dosing. Bold typeset indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 178
3.17 Fema1e dose group protein ranges at day 414 post dosing. Bold typeset indicates significant differences. The numbers are shown as a percentage of the total of the peaks quantified ... 183
A.1 Oil Spill Non-Governmental Organizations (NGO's) based in North America ... 223 B.1 Summary of statistical analyses (ANOV A) for serum proteins in male
and female mallards at sampling time- da ys 1 and 2 ... 251 B.2 Summary of statistical analyses (ANOV A) for serum proteins in female
mallards at sampling time- da ys 3 and 4 ... 252 B.3 Summary of statistical analyses (ANOV A) for serum proteins in male
mallards at sampling time- days 6 and 7 ... 253 B.4 Summary of statistical analyses (ANOVA) for serum proteins in male
mallards at sampling time- da ys 19 and 20 ... 254 B.5 Summary of statistical analyses (ANOV A) for serum proteins at
sampling time -day 72- female mallards ... 255
B.6 Summary of statistical analyses (ANOVA) for serum proteins at sampling time -day 278- female mallards ... 256 B. 7 Summary of statistical analyses (ANOV A) for serum proteins at
sampling time -day 334 -male mallards ... 257 B.8 Summary of statistical analyses (ANOVA) for serum proteins at
Les techniques actuelles utilisées lors de la réhabilitation d'oiseaux mazoutés capturés lors d'un déversement de pétrole donnent des résultats avec un haut taux de réussite. Cependant, des études sur la survie des oiseaux après la réhabilitation suivant un déversement de pétrole, ont mis en doute leur taux de survie après leur remise en liberté (Sharpe, 1996; Goldsworthy et al, 2000; Newman et al, 2003.). L'électrophérogramrne de protéinés est une méthode couramment utilisée pour aider à évaluer de nombreuses fonctions physiologiques au sein de la médecine humaine et vétérinaire. Au moyen de 1 'utilisation de l'électrophorèse capillaire dans le développement de l'électrophérogramrne, une technique qui nécessite une taille d'échantillon très faible tout en donnant un grand nombre d'informations, des profils ont été développés pour des canards colverts (Anas platyrynchos). Des canards colverts se sont fait administrer par voie orale des faibles, moyens ou élevés volumes de pétrole brut léger Brent en volumes similaires à ceux des déversements d'hydrocarbures, sous la présence d'un groupe de contrôle positif. Les traitements ont eu lieu en juillet et les oiseaux ont été suivis pendant un an. Des échantillons de sang ont été recueillis au cours de l'année englobant le cycle de reproduction annuelle, et des électrophérogramrnes de protéines ont été développés. Cela a permis l'identification à la fois positive (augmentation) et négative (diminution) des protéines liées à la réponse du corps à un stress de la phase aigüe (immédiatement après l'évènement) à la phase chronique. La quantification relative de ces pics de protéines et leurs réponses ont lié des protéines individuelles avec d'importants résultats biologiques pour les groupes exposés au pétrole. Les profils des protéines liées à ce résultat ont également été réparties par sexe. D'autres résultats ont montrés les tendances vers une liaison paire réduite, la baisse de fertilité, l'augmentation de la production d'œufs et des niveaux accrus d'agitation. La prolactine, une hormone quantifiée à plusieurs reprises au cours de la recherche, a augmenté de façon significative chez les femelles recevant la dose de l'huile élevée lors de la phase post reproductive en phase avec tous les autres oiseaux étudiés. L'utilisation de profils protéiques et la quantification des protéines individuelles offrent de précieux renseignements sur l'issue possible des oiseaux affectés par le pétrole par rapport aux oiseaux normaux et en santé. Cette étude a abouti à l'élaboration d'un autre outil de triage afin d'aider à l'évaluation du stress toxique à l'ingestion de l'huile.
Mots clés: paire liaison, électrophorèse capillaire, prolactine, déversement de pétrole, protéines de phase aigüe
Current techniques used during the rehabilitation of oiled birds captured during an oil spill have a high degree of success. However, studies exploring the survival of birds after oil spill rehabilitation have questioned their post-release survival. The protein electropherograrn is a commonly used method to aid in evaluating many physiological functions within human and veterinary medicine. Using capillary electrophoresis to develop the electropherograrn, a technique which requires a very small sarnple size while giving a great deal of information, protein profiles were developed for mal lards (Anas platyrynchos ). Mallards were dosed orally with either low, medium and high volumes of light crude Brent oil in volumes similar to oil spill exposures, and compared to a negative control group. Oil dosing was conducted in July and the birds were followed for one year. Blood samples were collected over the year encompassing the annual breeding cycle, and electropherograms were evaluated. This allowed for the identification of both positive (increased) and negative (decreased) protein responses during the acute phase (immediately following the event) to the chronic phase. Partial quantification (i.e. percentages) of these protein peaks; transthyretin, albumin, alpha 1, alpha 2, beta globulins and gamma globulins, along with two unidentified peaks, and their response served to link individual proteins with significant biological outcomes for the oil dose groups. The proteins linked to this outcome were also gender specifie. Other results included trends toward reduced pair bonding, lowered fertility, increased egg production, and increased agitation levels. The hormone prolactin was quantified at several times points over the course of the research and found to be ·significantly increased in the high oil dose females in the post reproductive phase as distinct from all other birds in the research. The use of protein profiles and the quantification of individual proteins offer valuable insight into the potential outcome for oil impacted birds when compared to normal birds. This study has contributed essential preliminary information leading towards the development of a new triage tool to aid in the evaluation oftoxic stress from the ingestion of oil.
Key words: Capillary electrophoresis, pair bonding, prolactin, oil spill, acute phase pro teins
INTRODUCTION
The current approach to handling captured oil-exposed wild birds involves a multi-tiered rehabilitation process that is physiologically stressful for the birds. Once captured, birds are evaluated to decide if they are suitable candidates for rehabilitation. Those that are suitable are stabilized with fluids to restore them to normal hydration, and then fed. When stable, those that are oiled or have lost waterproofing are washed following the established criteria for water temperature, water hardness, non-taxie compounds such as methyloleate or canola ail can be used as a pre-treatrnent when needed to ·soften tarry petroleum products, and Dawn® dish detergent (Berg, 2003). This results in birds with cleaned feathers and restored water proofing. The next step in the rehabilitation process is to bouse the birds from days to weeks, to ensure that they are clean, healthy, and fully waterproofed. The return of birds to the wild is then dependent on the release site, which must either have recovered from the ail spill, or be a suitable distance from it to ensure the birds do not return and become re-oiled.
Survival data from historical and recent spills are sketchy; however, what data do exist indicate that these birds have a limited survival period after handling (Sharpe, 1996; Environment Canada, 1996; Schmidt, 1997; Newman et al., 2003) even though the rehabilitation of oiled birds appears to be useful in restoring sorne birds to releasable condition. For example, Tri-State Bird Rescue, Delaware, U.S.A. reports a release rate of between 45 and 90% depending on the species and weather conditions (persona! communication, L. Frink). Given the high cast of handling oiled birds, the lirnited
technical and human resources, and the fact that many birds are severely affected before undergoing the stressful cleaning procedure, the currently applied rehabilitation strategy merits improvement. If we could initially measure a biological pararneter or pararneters, which would predict long term health, then it would be possible to better choose the subsequent course of treatment, including euthanasia wh en appropriate.
The main objective of the present investigation was therefore to develop a technique that is practical and inexpensive to add to the evaluation criteria of the birds admitted during an oil spill response. Acute phase proteins were evaluated as possible predictors of long-term health status in birds. Acute phase proteins are well documented and characterized in the human and agriculturally-oriented veterinary literature. The induction of acute phase proteins has a known time line that can be followed from days to weeks. In the field, where sample collection and storage may present difficulties in maintaining appropriate storage temperatures, these proteins will remain stable even through temperature fluctuations. Since protein electrophoresis and analysis of many acute phase proteins are routine tests in hospital and veterinary laboratories, the oil spill response teams could rapidly arrange with a local hospital or veterinary laboratory to quickly run the assays.
The test species chosen for this research was the mallard (Anas platyrhyncos ), who se natural habitat frequently occurs where oil spills are most likely to be found. In North America, mallards are the most abundant duck species and are very tolerant of human activity (Drilling et al., 2002). Due to the mallard's tolerance of human handling,
stress-induced infections such as aspergillosis are less likely to develop (Tseng, 1999; Carrasco, 2001).
Both behaviour and reproduction were examined because they are essential to the survival of the rehabilitated individuals and populations. Additionally these parameters are measureable and quantifiable with references to normality in the literature (Caldwell and Comell, 1975).
CHAPITRE!
Oil, the basis for much of our economie and social development over the past century, has been a natural part of the environment since the start of its formation during the Carboniferous period. It has now become an integral part of our modem world. However, by its very nature we have difficulty in containing it, and limiting its impact once free of its containment.
1.1. Historical background about oil spills
As shipping evolved in the late 19th century from sail and steam to petrochemical-powered vessels, spills and oily waste became a common sight around harbors and coastlines. Oil was often deliberately spilled 'to calm the waters' and indeed the 30' vessel Detroit carried oil for this purpose on the frrst crossing of the Atlantic Ocean by a petrochemical driven boat during the surnmer of 1912, as reported in the newspaper the Detroit News of June 26t11, 1912. Little attention was given to the environmental impact, especially as these spills were generally small and the recognition of the damaging environmental effects of oil spills was yet to be quantified.
1.1.1. The first oil tanker spill
The crude oil from the wreck of the Torrey Canyon led to the first massive public response to oil spills and their effect on seabirds and the environment. At 8:50 a.m. on
the 18th ofFebruary, 1967 the Torrey Canyon grounded on the Seven Stones Reef, off the
western coast of Cornwall, England, ultimately spilling her whole cargo of 117,000 tons of Kuwaiti crude (Baker, 1976). Appeals for funding and help appeared in many public sources (British Birds, 1967). Many books and publications explored and analyzed the
disaster (Cowan, 1968; Petrow, 1968). Estimates of the number of birds killed by this
spill begin at 30,000 (Boume, 1968). However, evidence from mark and recapture
experiments, using dead birds released at the spill site and recovered on beaches, place this figure closer to 100,000 birds. Of the nearly 8,000 birds brought in alive, only 100
were still alive 6 weeks later (Petrow, 1968).
1.1.2. The media response to the plight of oiled birds
Since then, the media has captured the public's attention through the many images
depicting the plight of oiled birds. Often, the frrst view the public has of an oïl spill event is the photo on the front page of the newspaper, the television report or the in-depth
report in magazines such as the National Geographie (January, 1990) (Figure 1.1).
1.1.3. Oil responses -the Canadian situation
The Canadian Wildlife Service (CWS), as lead agency m the response to the
environrnental impact during an oil spill, also promotes awareness through its Hinterland
Who's Who pamphlet 'Oil pollution and birds' (1992). This bas been followed up further
with a po licy in 2000, detailing the role that CWS will play in the event of oiling of birds,
especially those species at risk (Environrnent Canada, 2000). The three main areas of response are;
1. Knowing and providing information on the migratory bird resource and species at
risk (under CWS jurisdiction) in the area of a spill (this includes damage assessment and restoration planning after the event).
2. Minirnizing the damage to birds by deterring unoiled birds from becoming oiled.
3. Ensuring the humane treatment of captured migratory birds and species at risk by
determining the appropriate response and treatrnent strategies, which may include
euthanasia, or cleaning and rehabilitation.
Thus, the need to mount an oil spill response is driven through public pressure, increased
knowledge of appropriate techniques and legislative policy. However, further
investigation needs to be undertaken to ens ure a thorough understanding of the impact of an oil spill on the avifauna and given the current state of knowledge, what is the most
1.2. Oil impacts on birds and the rehabilitation effort
1.2.1. Background
Oil impacts birds at many levels. Initially, plumage is fouled through contact with a slick or spill. In the early evolution of the event, the bird will try to clean and restore its water proofing. As the bird tries to preen the oil from its feathers it unfortunately spreads the oil over a greater percentage of its plumes and further jeopardizes its thermoregulatory ability (Jenssen and Ekker, 1991). During this period it is also inhaling any volatile components from the oil. During preening it ingests biologically significant quantities of the oil (Hartung, 1964). Experimentally, it bas been shown that a bird can preen 50% of the oil on its feathers in a five to six day period (Hartung, 1963), although much of this activity often ceases after three to four days as the bird begins to become dehydrated, emaciated and obviously ill (persona! communication L. Frink, Tri-State Bird Rescue). Leighton (1994) produced a report which touched on many aspects of the impact that oil bas on the avian body. In summary Leighton's report looks at the following factors: extemal contamination leading to loss of water-repellency, insulation and flight, embryo toxicity, systernic toxicity including depressed growth and emaciation, adrenal gland enlargement, perturbations in thyroid hormones and corticosterone, liver damage, osmoregulatory impacts, decreased absorption of nutrients, reproductive !osses, immune suppression and anaemia. This report is further supported by results of other researchers (Table 1.1 ).
Table 1.1 A summary of pathological fmdings reported in oiled birds submitted for pathological examination.
Oil type Species # ofbirds Pathology Reference
cru de common murres 13 weight Joss/emaciation Khan and Ryan, 1991
thick-bilJed murres 2 necrosis in li ver and duodenwn,
renal tubular degeneration
herring gulls ? increased thyroxine, Peakall etal., 1981 black guillemots corticosterone and ACTH•
Leach's petrels
herring gu!Js 24 weight Joss Peakall et al., 1985 herring gu!Js 24 hemolytic anaemia Leightop. et al., 1985 herring gulls 72 decreased growth Lee et al., 1985
hemoJytic anaemia raised Cytocbrome P-450
mixed origin mixed species 241 emaciation and Jauniaux et al., 1997 haemorrhagic
gastro-enteropathy
he avy rhinoceros auklets 44 weight loss Oka and Okuyama, 2000
a. Adrenocorticotropic ho1mone
Historically, oiled birds were often left to die or were killed, and the earliest efforts in oiled bird rehabilitation from records dating back to 1942 were dismal failures (Berkner, 1979; Newman et al., 2003). Many techniques have been tried, from the use of solvents and cleaning agents (Odham, 1971; Newman et al., 2003), to iron powder (Orbeil et al., 1999), and specially designed automated bird washing machines (Mazet et al., 2002).
1.2.2. Development of a professional wildlife rehabilitation response to oil
spills
With the establishment of maJor rehabilitation centers (International Bird Rescue Research Center, Long Beach, California, established in 1971 and Tri-State Bird Rescue and Research, Inc, Newark, Delaware, established in 1976) specializing in oil spill responses, the use of a multidisciplinary approach has improved basic bird care. Established guides (Tri-State guide, 1990; Beaulieu and Fitzgerald, 1996; Daigle and Darveau, 1995; Berg, 2003; Mazet, 2003) now include protocols for;
capture and handling to minimize further stress or injury, euthanasia criteria,
immediate care to minimize further impact of oil and oily substances,
warming or cooling ( depending on season and location) while bird stabilizes and begins to rehydrate,
washing the bird,
drying and re-establishing waterproofmg,
maintaining adequate diet and ensuring weight gain, releasing into a clean environment.
These guidelines are put into use and tailored to the spill, species involved, environment, and public and legislative pressures. Each spill is different, yet the response is still based on the same principles (Appendix 1).
From the early 1970's there have been improved survival rates for birds entering the rehabilitation facility, with up to 100% of the birds admitted being released (Edmondson, 1981; Tri-State reports, 2002; Mazet et al., 2002). Much of the scientific work of discovery has been charnpioned by the Oiled Wildlife Care Network (OWCN) which was founded in the wake of the Exxon V aidez oil spi li by legislative efforts in California (Newman et al., 2003). It is administered at the University of California, School of Veterinary Medicine, Davis, California, to promote scientific based recovery efforts and research for oiled wildlife. This team also attends and aids with oil spill responses (Table 1.2).
Severa! studies suggest that the rehabilitation process may be very successful for sorne species (Golightly et al., 2002). Western gulls followed after rehabilitation for oiling showed normal dispersal and survival compared to unoiled birds. Pelicans also tend to do weil after rehabilitation (Anderson et al., 1996). There bas been continued work aimed at ensuring that the protocols for the rehabilitation of oil-affected birds utilize the most efficient and safe methods to manage the patients (Jenssen and Ekker, 1989; White, 1991; Mazet et al., 2002).
Table 1.2 Oiled Wildlife Care Network spill responses in which more than 25 live birds were recovered and rehabilitated (Newman et al., 2003).
Name And Location Of Major Spills* Date of Spill # Birds
Clark Pipeline, Huntington Beach Oct 1996- Nov 1996 35
Cape Mohican, San Francisco Bay Oct 1996- Nov 1996 58
Ballona Creek, Long Beach Jan 1997-Feb 1997 160 Torch/Platform Irene, Lompoc Sept 1997 - Oct 1997 53
Monterey Mystery, Santa Cruz Oct 1997 - Nov 1997 505
Kure, Eureka November 1997 484
Pt. Reyes Mystery #1, Pt. Reyes Nov 1997-Dec 1997 303
Carson, Carson January 1998 153
Pt. Reyes Mystery #2, Pt. Reyes Dec 1997-Mar 1998 635
Command, San Mateo City . . Sept 1998-Nov 1998 76
Wintersburg Channel, Huntington Beach Dec 1998 50
Golden West, Huntington Beach Jan 1999- Feb 1999 35
Calloway Canal, Bakersfield June 1998 25
Stuyvesant, Eureka September 1999 644
Trona, Trona June & Sept 2000 29
Stockdale, Bakersfield Oct 1999 155
Luckenbach, Pt. Reyes to Monterey Nov 2001-Jan 2003 1,095
*
Overall, over 40 spill responses and more than 4,300 live birds collected for care.One other factor must also be noted when examining survival rates. Oil is not a simple product, but rather a mix of many compounds reflecting its origins and subsequent processing (Miller et al., 1982; Jokuty et al., 1989; Fingas, 2001). Thus each spill's survival success or failure rate must also be examined in light of the chemical properties of the oil itself (Hartung and Hunt, 1966).
1.2.3. Survival data in birds following oil spill exposure
Sharpe (1996) examined the band returns reported for birds banded in the course of
normal biological surveys (control birds) versus the band return data collected on birds
admitted to a rehabilitation facility for cleaning in the aftermath of an oil spill event
(Figure 1.2). The oiled birds, once cleaned and released, showed a significant decrease in
long term survival when compared to .the control birds. This led to concerns about the
value of rehabilitation techniques and the Jack of understanding of the real impact these
birds endure after such an event (Environment Canada, 1996; Anderson, 2000; Briggs, 1996; Briggs, 1997; Newman, 1999 and 2000).
Mean Days Survived After Release
1200 1000 o Western Grebe 800 Vl • Guillemot >-600 ro
0 o Velvet Seo ter
400 200 0
Non Oiled Oiled
Figure 1.2 The number of days for bands to be recovered between non-oiled birds (control) vs. oiled, rehabilitated and released birds (adapted from Sharpe, 1996).
Cleaning is a well-established process, and Sharpe (1996) noted that the data presented showed no difference in band return days in birds treated prior to the institution of the most efficacious oiled bird rehabilitation process compared to those techniques in practice before the ear1y 1990's. There are other issues involved, potentially those relating to the toxic impact of the petroleum products.
1.2.4. Oil toxicity in birds
Research on mallards and Pekin ducks (Anas peking) highlighted the impact that the
toxicity itself can have (Hartung and Hunt, 1966). The impacts resulting from the ingestion of various oils and oil products on these birds included lipid pneumonia, decreased red blood cells, hematocrit and hemoglobin levels, increased liver enzymes, nephritic changes and adrenal gland enlargement. Khan and Ryan (1991) described the condition of comrnon murres following a crude oil spill off the coast ofNewfoundland in January 1990. Post-mortem exarnination of birds showing evidence of oil contact revealed no fat stores, low hematocrit and intestinal necrosis. Sorne birds showed significant li ver changes including congestion, fatty de generation, and dissociation of the hepatocytes. All birds in this study had kidney function impacts including renal tubular degeneration and necrosis. Of the 15 birds presented for rehabilitation, only two were subsequently released. Oka and Okuyama (2000) found dead oiled rhinoceros auklets (Cerorhinca monocerata) with severe nutritional exhaustion, low liver weights and empty stomachs.
Anderson et al. (2000) followed the survival of American coots (Fulica americana) post
rehabilitation after being impacted by San Joaquin crude oil. Decreased survival, failure to regain normal body condition and behavioural changes, including increased preening activity, were all seen in this group of oil-impacted birds.
Fry and Addiego (1987) explored the health parameters of severa! species of seabirds following two oil spills in the central California region. Their findings indicated perturbations within the serum protein profiles consistent with an acute phase response. The other factor noted was a hemolytic anemia which in sorne cases was severe (1 0% red
blood cell loss ). Initially the raised total prote in levels were thought to be indications of dehydration, however, rehydration therapy failed to change them. Further investigation of the protein profiles of common murres indicated a loss of the pre-alburnin peak, with raised levels of alpha, beta and gamma globulin proteins.
1.2.5. Impacts of oil spills in birds at the population level
Trying to evaluate the real impact of a spill presents many problems. Surveying birds to
gain an understanding of population status is a useful technique, but one that needs to take into consideration geographical and temporal issues which may also affect assessment of true population levels. Once these issues were addressed, Esler et al. (2000,
2002) concluded that by 1997, the harlequin duck (Histrionicus histrionicus) population
impacted by the 1989 Exxon Valdez spill still had not fully recovered. The population had not reached its former levels nine years after the spill, a result in contrast to a
cornmonly held belief that oil spills, while impacting birds initially, have little long term impact at a population level. Golet et al. (2002) found a similar result in surveying the pigeon guillemot (Cepphus columba) populations in this region. These differences were also seen in sorne blood biochemistry values between birds from non-oiled and oiled areas. These included hepatic cytochrome P4501A (CYPlA), aspartate arninotransferase (AST), and lactate dehydrogenase (LDH) enzymes, which all remained elevated when compared to pigeon guillemots from non-oiled regions.
1.2.6. Impacts of oil spills on reproduction in birds
Following the rehabilitation of oiled brown pelicans (Pelecanus occidentalis), observations and band returns indicated that these birds had not on1y significantly lowered survival rates, but those that survived showed no breeding activity (Anderson et
al., 1996). Of interest, the se bll:ds did not return to the breeding colony during the
reproductive period. In Magellanic penguins (Spheniscus magellanicus) a similar picture emerged in birds that were oiled and rehabilitated (Fowler et al., 1995) although, those birds that did return to the breeding colony and remained paired were able to produce chicks. Thus there is compelling evidence that ingested petroleum products cause reproductive impairment even when rehabilitation has been successful.
Research into the reproductive aspects of birds impacted by oil shows sorne significant changes in hormonally-based processes. Ovarian development is delayed and male reproductive abilities are depressed when mallards are fed oil-laced food (Cavanaugh and Holmes, 1982, 1987). Sorne ofthese impacts may also be related to low plasma prolactin
concentrations (Cavanaugh et al., 1983) as well as depressed levels of estradiol and
progesterone (Cavanaugh and Holrnes, 1982). Peakall et al. (1981) noted elevations in
plasma corticosterone and thyroxine levels in nestling herring gulls and black guillemots
after one dose of either a crude oil or its aromatic fraction. The corticosterone level
responded imrnediately to the dose event while the thyroxine levels were raised sorne six
days later. This increase persisted for two weeks. Further work by Peakall et al. (1985)
confirmed earlier results and highlighted the impact oil ingestion has on weight loss.
Following the Prestige oil spill off the coast ofGalicia, Spain, in 2003, the European shag
(Phalacrocorax aristotelis) colonies impacted by the oil were monitored for reproductive
perturbations. When compared to non-impacted colonies, those colonies influenced by
the spill showed a 50% reduction in breeding activity 01 elando et al., 2005). It is unclear
whether this can be attributed to sublethal oil exposure or low food availability in the
wake of damage from the spill.
1.2.7. Immune function impacts in oiled birds
Immune suppression of seabirds undergoing rehabilitation is always an issue. The stress
of the rehabilitation process can induce secondary disease invaders, which contribute to
higher mortality and morbidity (Mazet et al., 2002). One area which may be overlooked
is the role that the petrochemicals themselves play in inducing immune function stress
(Briggs et al., 1996, 1997). The mechanisms of imrnune-mediated stress may also
seroprevalence of avian malaria seen injackass penguins (Spheniscus demersus) in South Africa (Graczyk et al., 1995) that had been oiled and rehabilitated.
1.2.8. Impacts of oil in young birds
Ducklings are also vulnerable to the impact of ingestion of oil. Mallard ducklings were fed No. 2 fuel oil, a refmed petro1eum product, during the frrst eight weeks of development (Szaro et al., 1981 ). Growth was depressed in the birds with diets containing 5% of the petroleum product. Liver hypertrophy and splenic atrophy were noted along with evidence of renal damage. Also noted were sorne behavioural changes in those ducklings.
1.2.9. Limitations to oiled bird rehabilitation
The impact of petroleum products both on and in the avian body often leads to the death of the bird. However, once an oil spill response is mounted, those birds entering the rehabilitation process have a high probability of release at a later date. In major spill events, rehabilitation priority is often given to species of concem and those individuals with minimal secondary problems, such as fractures and wounds. Even if small numbers of oiled birds are captured, triage ensures that only those individuals that are likely to benefit most from the rehabilitation process are admitted. At present, triage is often based on overall condition and sorne common biochernical and hematological tests including a hematocrit (hct) to measure packed cell volume (pcv) and the proportion of the buffy coat
(white celllayer), and total solids as measured by a refractometer. These procedures are described in Appendix A, a case study of the rehabilitation process based at the Tri-State Bird Rescue and Research facility, Fort George, Louisiana, during the Deepwater Horizon oil spill, in the Gulf of Mexico, off the coast of Louisiana in 201 0.
However, the present triag·e protocols may not be enough to ensure that the rehabilitated birds are capable of long term survival and successful reproduction. Therefore other parameters should be evaluated for inclusion in the evaluation process during the admission of oiled birds. The changes in the acute phase proteins may be the best option for ensuring the rehabilitation of only those birds that are able to return to their lives in the wild as expected for their species, and reproduce successfully.
1.3. Acute Phase Proteins in the context of oil spill responses
An oil spill event where birds are oiled is a potentially toxic assault. Physiological responses result in difficulties linked to thermoregulation, nutrition and hydration. The resulting shift in homeostasis would be expected to induce an acute phase response with the attendant changes in levels of acute phase proteins.
1.3.1. An overview of the acute phase response and the subsequent induction of acute phase proteins.
The use of human acute phase proteins as markers in a clinical study was frrst described in 1930 by Tillet and Francis. The induction or suppression of acute phase proteins is a
part of the organism's attempt to restore homeostasis (Heinrich et al., 1990). This
complex response (Figure 1.3) to an assault on the body, whether it is from an infection,
trauma, neoplasia, stress or inflammation, can result in a wide range of change in levels
of sorne of the proteins, specifically those known as the acute phase proteins (Heinrich et
al., 1990). The inflarnmatory process and subsequent acute phase response requires
precise regulation to ensure an appropriate response, which is not compounded by
immaturity, immunosuppressive disease, genetics or stress (Klasing, 1998). The acute phase proteins arise from stimulation of the hepatocytes in the li ver (Miller et al., 1951) by cytokines including interleukin (IL) 6-~, interleukin (IL) 1 and tumor necrosis factor-alpha (TNF-a) (Figure 1.4).
